Methods of lyophilization or freeze drying for preserving liposomes containing biologically active molecules and for bacterial cultures are known. Preparation of pharmaceutical tablets using lyophilizing procedures also are known. In U.S. Pat. No. 4,678,812, a method preparing tablets useful in diagnostic applications utilizing trehalose as an excipient and stabilizer is disclosed. U.S. Pat. No. 4,712,310 teaches a method of preparing tablets useful as reagent carriers for diagnostic assays.
Methods of encapsulating materials such as drugs, nucleic acids, proteins, reporter molecules, enzymes and the like into liposomes are taught in prior art patents. Examples of such patents are U.S. Pat. Nos. 4,515,736 and 4,411,894. U.S. Pat. Nos. 3,261,761, 4,206,200 and 4,246,349 teach lyophilizing procedures for bacteria. In this same study area is the published European Patent Application No. 0 259 739, published Mar. 16, 1988, entitled "Improved Stability of freeze-dried cultures".
The prior art identified addresses the technique of using sugars in their lyophilization applications for a stabilizing function. The sugar trehalose also is mentioned as being useful for this function. In International Application published under the PCT, No. WO 86/03938 published July 17, 1986, there is disclosed use of trehalose as a preserving agent, both inside the liposomes and externally, in solution, during freeze-drying. This use of trehalose both externally and internally of liposomes is the preferred embodiment noted.
None of the prior art technology described undertakes to preserve mammalian cells and particularly human cells. Human cells such as blood cells are distinctive in their membrane structure as compared to the membrane structure of bacteria or liposomes. In the case of bacteria, the cell wall is a very thick non-lipid wall whose function is to protect the cell fluid contents from such adverse effects as heat, osmotic pressure changes and even freeze-drying in the absence of a preserving agent, such as a sugar. Thus, many strains of bacteria can withstand the stresses of freezing and drying in the absence of a unique preserving protective or coating because of their rigid cell wall.
In the case of liposomes, the fluid space is enclosed by unilamellar or multilamellar lipid vesicles. The walls of the vesicles are formed by a biomolecular layer of one or more lipid components having polar heads and non-polar tails. In an aqueous solution, the polar heads of one layer orient outwardly to extend into the surrounding medium, and the non-polar tail portions of the lipids associate with each other, thus providing a polar surface and a non-polar core in the wall of the vesicle. Unilamellar liposomes have one biomolecular layer whereas multilamellar liposomes generally have a plurality of substantially concentric biomolecular layers. Thus, the liposome is a spherical microstructure formed when mixtures of phospholipids with or without steroids are dispersed in aqueous solutions. They are envisioned as useful for encapsulating an agent for recognized delivery to an in vivo site.
The liposome cell wall is an artificial bi-lipid membrane as compared to that of a human cell, for instance. The composition of a human cell membrane is much more complex. In addition to a bi-lipid membrane structure, the human cell membrane has many additional protein and glycoprotein molecules intercolated into its lipid bi-layer. These proteinaceous molecules are of particular interest in that they provide cell surface antigens or determinant sites not present in an artificial liposome. The investigation of human cell surface markers is the crux of immunoassays and other hematological measurements relating to human blood cells. The same distinction over liposomes is apparent in use of hybridoma cell lines and monoclonal antibodies. Another distinction to keep in mind is the difference between the complex fluid contents of a mammalian cell and that of a liposome, for instance. In the instance of a human blood cell, the cell exists only in an isotonic fluid medium which is not the case for liposome which may exist in any medium, including non-isotonic solutions.
The lyophilizing method embodying the invention is unique in that mammalian cells are lyophilized without adversely changing the morphology of the cell and without rendering the cell membrane permeable after conjugation to a probe or marker for its application in an assay, for instance, when constituted as a control cell. Further, said lyophilizing method functions to stabilize the proteinacious structure of the cell membrane and its orientation within the cell wall so that the membrane surface protein markers are not destroyed. Considering the known fragile character of the cell membrane of mammalian cells, the results achieved by the lyophilization method of the herein invention are believed to have been unexpected and suprisingly effective in terms of stability and shelf life of the lyophilized mammalian cell achieved as well as retaining physiological functional characteristics after rehydration as compared to fresh cells.
In practicing the method embodying the invention, the sugar trehalose in an isotonic solution is utilized as a preservative or protective coating on the exterior surface only of a mammalian cell. Coating the interior surface of the cell membrane would not be feasible. The characteristics of excellent stability and longevity of shelf life achieved enables the invention to be applied to producing control cells for immunological assays, for use in assuring proper sample preparation for flow cytometric analyses of mammalian cells, for blood cell counting, sizing and analysis of subsets of blood cell constituents, such as red cells and white cells and in DNA analysis for a variety of cell types. Thus, various types of cells can be lyophilized and thereafter reconstituted for successful biological cell analysis.
In Stefi Weisburd, "Death Defying Dehydration", Science News, Vol. 133, No. 7, pp. 97-112, Feb. 13, 1988, there is described a process for freeze-drying liposomes using trehalose as described in said International Application under PCT No. WO86/03938. The publication described research conducted with micro-organisms and showing that trehalose, a low molecular weight sugar, was produced in high concentrations in these micro-organisms as they dehydrated. It was considered that trehalose acted as a lipid stabilizer to increase the stability of the artificial cell-membranes or liposomes. The publication cautions that trehalose may not be a useful additive in all situations and that the toxicology of trehalose should be noted.
The need for accurate and reliable control media which can be assayed concurrently with the test samples to realize proper sample preparatory technique and laboratory test instrument function is a continuing one. In flow cytometric analysis, no established controls are available except fluorescent beads or microspheres which are used to set the flow cytometer operating parameters. The most desirable control substance would be one that could control for reagents utilized and sample preparatory technique along with proper instrument function. Thus, for flow cytometric analyses, preserved mammalian cells expressing the required antigenic determinant or determinants to be assayed along with cells of the test sample would present an optimal control medium. Previously determined values for the control cells could be compared with test results obtained by the researcher.
The nature of flow cytometric analysis for all surface antigens requires a preparatory method for the control cells which preserves cellular light scattering patterns and membrane integrity with minimal damage to the cell surface antigens being studied. The best basis of comparison for these factors would be fresh cells having the known properties to be studied. However, shipment and storage of fresh control cells in a laboratory environment would be impractical from a commercial supply standpoint.
Thus, preserved mammalian cells such as by lyophilization which could be shipped and stored for a desirable shelf life period and reconstituted or rehydrated at the time of use while retaining the necessary characteristics for its use would provide a desirable control for flow cytometric analyses.
Such lyophilized mammalian control cells also would be suitable for use with hematology analysis instruments, such as the COULTER COUNTER.RTM. blood cell analyzer marketed by Coulter Electronics, Inc. of Hialeah, Fla., a wholly owned subsidiary of Coulter Corporation, assignee of record.
Applicants believe that freeze-drying of mammalian cells for use as control media has not been successfully achieved for several reasons. For instance, the freezing parameters adversely affects proteinacious molecules in such cells and can destroy all membrane integrity.
Experimental work utilizing a variety of sugars in combination with DMSO or glycerol to counter the adverse effects of intra-cellular freezing were undertaken. Also, sugars alone were utilized in the lyophilizing procedure wherein the sugar was added to the lyophilization carrier solution, such as standard normal goat serum, for the cells in suspension during the freeze down and lyophilization procedure employed. Various dilutions of sugars with and without DMSO, phosphate buffered albumin (PBA), fetal calf serum, Human AB Serum and Normal Goat Serum were tried.
Experiments using sucrose, taurine and glucose, and DMSO with sugar as a carrier all were unacceptable in results achieved. For example, incidents of decrease in specific fluorescence for some antigens relative to fresh cells, unacceptable light scatter and specific fluorescence characteristics were determined when fructose was used. The treatment of cells using such sugars still provided unsatisfactory lyophilized cells such that the cells were not properly prepared to prevent major damage during freeze down. Such damage was expressed in molecular change in cell membrane proteins, such as conformation alterations which could affect antigen expression. Cryoprotectorants are needed to routinely prevent such damage that occurs as a result of increased salt concentrations and crystal formation during freezing of cells. Such cryoprotectorants as DMSO and glycerol, as well as other serums, were found to be unsuccessful for lyophilization of mammalian cells by applicants.
The use of normal goat serum as a matrix or carrier in which the cells are suspended during freeze-down and lyophilization did not provide optimal cell surface marker survival. It was believed that the protein in the serum did not protect the cell membrane from oxidation and other disruption. Trehalose used only and in mixtures with normal goat serum and albumin also proved to be unsuccessful in solving the problems alluded to herein.
Thereafter, experiments were conducted using the sugar trehalose as part of several carrier formulas including mixtures with normal goat serum and albumin without achieving an acceptable lyophilized control cell medium. Thus, the use of sugars, including trehalose for stabilizing membrane lipids of cells during lyophilization proved unsuccessful.
The method embodying the invention which proved successful for providing acceptable lyophilized mammalian cells comprises utilizing the sugar trehalose in an isotonic solution for preparing the cells for the rigor of the freeze-down and lyophilizing protocols. The lyophilized control cells produced by the inventive method included hybridomas or cell lines and peripheral blood mononuclear cells which retained optimal characteristics for use in flow cytometer analyses. An optimal trehalose concentration in isotonic solution also was determined with trehalose being the most acceptable sugar for use in the inventive method. The invention includes the lyophilized mammalian control cells and other human cells capable of being produced by said lyophilization method.